Revision as of 13:59, 15 February 2013

Contents

Update for everyone

As explained in pre-lab lecture, we were remiss (mainly me, Agi!) in not providing you with the best sequence information for V corneae. Moreover, we told you to shoot for a Tm of 58 °C instead of a Tm of 63 °C (Ta of 58 °C).

Below, we have listed modified primer sequences (just under your originals) that match the Broad sequence. An ApE file of this sequence is linked [Vcorneae_SSU-Broad-direct.gb here].in progress

As a default, we will order these primers on Tuesday and Wednesday for arrival on Thursday and Friday, and run two separate PCRs: one at Ta = 58 °C and one at Ta = 53 °C.

However, if you would like to revise your primers to a Tm of 63 °C, we will accept these new designs through Monday at 9 pm for T/R section and Tuesday at 9 pm for W/F section.

T/R section

Please sign up for one of the design challenges -- four teams per challenge -- right away.

When you have finished, post your primer sequences (from 5' to 3') so that they can be ordered.

I will name your primers by the approximate base-pair that they target in V corneae or E hellem, so please share this information as well.

Sensitivity

Team color

Forward (F) primer

Reverse (R) primer

Target site (e.g. bp 60), F

Target side (e.g., bp 300), R

Is bp # for VC or EH? Which gene?

Grey (Platinum) :)

CAT GCT TGC CAA CAC AGPT so unchanged

GAA TGG TCT GGT TGC TTCPT so unchanged

bp 9

bp 251

E. Hellem: polar tube protein gene

Green

AGG TTG ATT CTG CCT GACfine

CGG AAC CAA ACC CTG ATCGG TAC AAA ACC CTA AT

bp 5

bp 236

VC: SSU rRNA

Pink

ttctgcctgacgtagatgfine

cctctccggaaccaaacCCT CTC CGG TAC AAA AC

bp 12

bp 226

VC

Yellow

CTGACGTGGATGCTATTCctg acg tag atg cta gtc

CCGTCAACCGTCACTGTCfine

bp 18

bp 199

VC small subunit ribosomal RNA gene

Specificity

Team Color

Forward primer

Reverse primer

Target site (e.g. bp 60), F

Target side (e.g., bp 300), R

Is bp # for VC or EH? Which gene?

Purple

5' GCG ATG ATT TAG TCT GGC 3'

5’ CAA TTT CCA ACG ACC ATG C 3’

bp 93, F

bp 879, R

VC, SSU rRNA

Red

GGT TGA TTC TGC CTG ACG TGG

CCT TCG TCC TTC ATC ACC ACA A

bp 6, F

bp 588, R

VC, SSU RNA

Orange

5'cgt agt cat aga agg gca aag ag3'

5' ctc tct ctc tac ctg cta ttg ta 3'

441

1005

V. corneae TV4/TV5 SSU rRNA

Blue

CCGCAATCAGGGACGAA

GTCAACGTCACTGTCTTGG

60

195

V. corneae TV4/TV5 SSU rRNA

W/F section

Please sign up for one of the design challenges -- four teams per challenge -- right away.

When you have finished, post your primer sequences (from 5' to 3') so that they can be ordered.

I will name your primers by the approximate base-pair that they target in V corneae or E hellem, so please share this information as well.